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넙치 3가지 타입 인지질가수분해효소(PLC-δ1)의 세포 내 위치 및 이동

Cellular Localization and Translocation of Duplication and Alternative Splicing Variants of Olive Flounder Phospholipase C-δ1

  • Kim, Na Young (Pathology Research Division, National Institute of Fisheries Science (NIFS)) ;
  • Kim, Moo-Sang (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Jung, Sung Hee (Pathology Research Division, National Institute of Fisheries Science (NIFS)) ;
  • Kim, Myoung Sug (Pathology Research Division, National Institute of Fisheries Science (NIFS)) ;
  • Cho, Mi Young (Pathology Research Division, National Institute of Fisheries Science (NIFS)) ;
  • Chung, oon Ki (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Ahn, Sang Jung (Fisheries R&D Management Center, Korea Institute of Marine Science & Technology Promotion (KIMST))
  • 투고 : 2017.09.15
  • 심사 : 2017.10.26
  • 발행 : 2017.11.30

초록

본 연구의 목적은 넙치 인지질가수분해효소(PLC-${\delta}1$) 3가지 타입의 세포내 특성을 규명하고자 하였다. 일반적으로 인지질가수분해효소(PLC)의 신호전달경로는 핵, 세포막, 세포질에 분포한다고 알려져 있으나, 핵내 위치 메커니즘은 여전히 불분명하다. PoPLC-${\delta}1A$, PoPLC-${\delta}1B$ (Sf)과 PoPLC-${\delta}1B$ (Lf)의 3타입의 유전자들은 각각 핵위치 신호(NLS)와 핵방출서열(NES)을 포함하고 있다. 본 연구에서는, 넙치 3가지 타입 인지질가수분해효소(PLC-${\delta}1$)의 세포내 위치이동 메커니즘 분석을 위해 GFP 벡터에 유전자를 삽입하여 ionomycin과 thasogargin처리 후 세포위치와 이동양상을 공초점 레이저 주사현미경으로 관찰하였다. PoPLC-${\delta}1A$는 PoPLC-${\delta}1B$ (Lf)와 PoPLC-${\delta}1B$ (Sf)가 원형질막에 국한되어 분포할때 세포질과 세포막보다 세포 소기관에 분포되어 있었다. PoPLC-${\delta}1B$ (Lf) 및 PoPLC-${\delta}1$ (Sf)이 핵 세포질내 이동양상을 보이지 않을 때, PoPLC-${\delta}1A$은 ionomycin과 thapsigargin 처리에 의해 핵 내에 축적되는 양상을 나타냈다. 이런 결과는 손상되지 않은 기능적 NES 서열을 포함하는 PoPLC-${\delta}1A$가 어류에서 핵 세포질 내 왕복 및 이동의 주된 역할을 한다는 것을 보여주고 있다.

The purpose of this study was to investigate the cellular characterization of phospholipase C-${\delta}1$ in olive flounders (Paralichthys olivaceus). In general, phospholipase C signaling pathways are distributed in nuclei at plasma membranes and in cytoplasms, although the pathways' nuclear localization mechanisms are unclear. P. olivaceus duplicates type-A PoPLC-${\delta}1$ (PoPLC-${\delta}1A$), which has a high similarity to the human isoform PLC-${\delta}$; type-B PoPLC-${\delta}1$ (PoPLC-${\delta}1B$ [Sf]), which has a low similarity to the human isoform PLC-${\delta}$ and the alternative splice variant PoPLC-${\delta}1B$ (Lf), which has a nuclear localization signal (NLS) and a nuclear export signal (NES) for nuclear imports and exports, respectively. This study confirmed the effects of the cellular localization and translocation of GFP-tagged PoPLC-${\delta}1A$, PoPLC-${\delta}1B$ (Sf) and PoPLC-${\delta}1B$ (Lf). It administered treatments of $Ca^{2+}$ ionophore ionomycin and endoplasmic reticulum (ER)-$Ca^{2+}$ pump inhibitor thapsigargin to hirame natural-embryo (HINAE) cells. A laser-scanning confocal microscope was used. GFP-tagged PoPLC-${\delta}1A$ was distributed to the cellular organelles, rather than to the cytoplasms and cytomembranes, when PoPLC-${\delta}1B$ (Lf) and PoPLC-${\delta}1B$ (Sf) were localized at the plasma membranes. The treatments of ionomycin and thapsigargin showed the accumulation of PoPLC-${\delta}1A$ in the nuclei when PoPLC-${\delta}1B$ (Lf) nucleocytoplasmic shuttling and PoPLC-${\delta}1B$ (Sf) nucleocytoplasmic shuttling were not observed. The results were the first evidence that PoPLC-${\delta}1A$, which contains functional, intact NES sequences, has a main role in nucleocytoplasmic shuttling and translocation in fish.

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참고문헌

  1. Caprini, M., Gomis, A., Cabedo, H., Planells-Cases, R., Belmonte, C., Viana, F. and Ferrer-Montiel, A. 2003. GAP43 stimulates inositol triphosphate-mediated calcium release in response to hypotonicity. EMBO J. 22, 3004-3014. https://doi.org/10.1093/emboj/cdg294
  2. Dingwall, C. and Laskey, R.A. 1991. Nuclear targeting sequences-A consensus? Trends. Biochem. Sci. 16, 478-481. https://doi.org/10.1016/0968-0004(91)90184-W
  3. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. and Nishida, E. 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308-311. https://doi.org/10.1038/36894
  4. Ichinohe, M., Nakamura, Y., Sai, K., Nakahara, M., Yamaguchi, H. and Fukami, K. 2007. Lack of phospholipase C delta1 induces skin inflammation. Biochem. Biophys. Res. Commun. 356, 912-918. https://doi.org/10.1016/j.bbrc.2007.03.082
  5. Irino, Y., Cho, H., Nakamura, Y., Nakahara, M., Furutani, M., Suh, P. G., Takenawa, T. and Fukami, K. 2004. Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4. Biochem. Biophys. Res. Commun. 320, 537-543. https://doi.org/10.1016/j.bbrc.2004.05.206
  6. Katan, M. 1998. Families of phosphoinositide-specific phospholipase C: structure and function. Biochim. Biophys. Acta. 1436, 5-17. https://doi.org/10.1016/S0005-2760(98)00125-8
  7. Kato, H., Fukami, K., Shibasaki, F., Homma, Y. and Takenawa, T. 1992. Enhancement of phospholipase C delta 1 activity in the aortas of spontaneously hypertensive rats. J. Biol. Chem. 267, 6483-6487.
  8. Kim, N. Y., Kim, M. S., Ahn, S. J., Seo, J. S., Bak, H. J., Kim, B. S., Jo, H. I., Jang, H. Y., Jo, H. S., Lee, H. H. and Chung, J. K. 2013. Functional analysis of duplicated genes and N- terminal splice variant of phospholipase $C-{\delta}1$ in Paralichthys olivaceus. Comp. Biochem. Physiol. B. 165, 201-210. https://doi.org/10.1016/j.cbpb.2013.04.005
  9. Nakamura, Y., Fukami, K., Yu, H., Takenaka, K., Kataoka, Y., Shirakata, Y., Nishikawa, S., Hashimoto, K., Yoshida, N. and Takenawa, T. 2003. Phospholipase C ${\delta}1$ is required for skin stem cell lineage commitment. EMBO J. 22, 2981-2991. https://doi.org/10.1093/emboj/cdg302
  10. Nakamura, Y., Hamada, Y., Fujiwara, T., Enomoto, H., Hiroe, T., Tanaka, S., Nose, M., Nakahara, M., Yoshida, N., Takenawa, T. and Fukami, K. 2005. Phospholipase C-delta1 and -delta3 are essential in the trophoblast for placental development. Mol. Cell. Biol. 25, 10979-10988. https://doi.org/10.1128/MCB.25.24.10979-10988.2005
  11. Okada, M., Fujii, M., Yamaga, M., Sugimoto, H., Sadano, H., Osumi, T., Kamata, H., Hirata, H. and Yagisawa, H. 2002. Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C ${\delta}1$. Genes Cells 7, 985-996. https://doi.org/10.1046/j.1365-2443.2002.00577.x
  12. Okada, M., Ishimoto, T., Naito, Y., Hirata, H. and Yagisawa, H. 2005. Phospholipase $C{\delta}1$ associates with importin ${\beta}1$ and translocates into the nucleus in a $Ca^{2+}$-dependent manner. FEBS Lett. 579, 4949-4954. https://doi.org/10.1016/j.febslet.2005.07.082
  13. Rhee, S. G. 2001. Regulation of phosphoinositide-specific phospholipase C. Annu. Rev. Biochem. 70, 281-312. https://doi.org/10.1146/annurev.biochem.70.1.281
  14. Shimohama, S., Homma, Y., Suenaga, T., Fujimoto, S., Taniguchi, T., Araki, W., Yamaoka, Y., Takenawa, T. and Kimura, J. 1991. Aberrant accumulation of phospholipase $C-{\delta}$ in Alzheimer brains. Am. J. Pathol. 139, 737-742.
  15. Stallings, J. D., Tall, E. G., Pentyala, S. and Rebecchi, M. J. 2005. Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 280, 22060-22069. https://doi.org/10.1074/jbc.M413813200
  16. Stallings, J. D., Zeng, Y. X., Narvaez, F. and Rebecchi, M. J. 2008. Phospholipase C-delta1 is linked to proliferation, DNA synthesis and cyclin E levels. J. Biol. Chem. 283, 13992-14001. https://doi.org/10.1074/jbc.M800752200
  17. Stewart, A. J., Mukherjee, J., Roberts, S. J., Lester, D. and Farquharson, C. 2005. Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes. Int. J. Mol. Med. 15, 117-121.
  18. Yagisawa, H., Okada, M., Naito, Y., Sasaki, K., Yamaga, M. and Fujii, M. 2006. Coordinated intracellular translocation of phosphoinositide-specific phospholipase $C-{\delta}$ with the cell cycle. Biochim. Biophys. Acta. 1761, 522-534. https://doi.org/10.1016/j.bbalip.2006.02.016
  19. Yamaga, M., Fujii, M., Kamata, H., Hirata, H. and Yagisawa, H. 1999. Phospholipase $C-{\delta}1$ contains a functional nuclear export signal sequence. J. Biol. Chem. 274, 28537-28541. https://doi.org/10.1074/jbc.274.40.28537